Pokrishevsky Edward, Hong Ran Ha, Mackenzie Ian R, Cashman Neil R
Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
PLoS One. 2017 Sep 6;12(9):e0184384. doi: 10.1371/journal.pone.0184384. eCollection 2017.
Mutant Cu/Zn superoxide dismutase (SOD1) can confer its misfolding on wild-type SOD1 in living cells; the propagation of misfolding can also be transmitted between cells in vitro. Recent studies identified fluorescently-tagged SOD1G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, in vitro and in vivo. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1. We observed that human spinal cord homogenates prepared from SOD1 familial ALS (FALS) can induce significantly more intracellular reporter protein aggregation than spinal cord homogenates from sporadic ALS, Alzheimer's disease, multiple system atrophy or healthy control individuals. We also determined that the induction of reporter protein aggregation by SOD1-FALS tissue homogenates can be attenuated by incubating the cells with the SOD1 misfolding-specific antibody 3H1, or the small molecule 5-fluorouridine. Our study further implicates SOD1 as the seeding particle responsible for the spread of SOD1-FALS neurodegeneration from its initial onset site(s), and demonstrates two potential therapeutic strategies for SOD1-mediated disease. This work also comprises a medium-throughput cell-based platform of screening potential therapeutics to attenuate propagated aggregation of SOD1.
突变型铜锌超氧化物歧化酶（SOD1）可在活细胞中将其错误折叠传递给野生型SOD1；错误折叠的传播在体外细胞间也能发生。最近的研究将荧光标记的SOD1G85R鉴定为一种混杂底物，在体外和体内极易被多种模板诱导聚集。在此，我们利用了几种在SOD1中带有G37R、G85R或G93A突变的SOD1-GFP报告蛋白。我们观察到，与散发性肌萎缩侧索硬化症（ALS）、阿尔茨海默病、多系统萎缩症患者或健康对照个体的脊髓匀浆相比，取自SOD1家族性ALS（FALS）患者的人类脊髓匀浆能显著诱导更多的细胞内报告蛋白聚集。我们还确定，用SOD1错误折叠特异性抗体3H1或小分子5-氟尿苷处理细胞，可减弱SOD1-FALS组织匀浆对报告蛋白聚集的诱导作用。我们的研究进一步表明，SOD1是导致SOD1-FALS神经退行性变从其初始发病部位扩散的种子颗粒，并展示了两种针对SOD1介导疾病的潜在治疗策略。这项工作还构建了一个基于细胞的中通量平台，用于筛选潜在疗法以减弱SOD1的传播性聚集。
